Periodontal Disease Progression in Type II Non-Insulin-Dependent Diabetes Mellitus Patients (NIDDM). Part II - Microbiological Analysis Using the BANA Test

 
Arthur B. NOVAES Jr.1
Ferney GONZALEZ Gutierrez2
Marcio Fernando de MORAES GRISI3
Arthur B. NOVAES3
 
1Faculdade de Odontologia, Universidade de Rio de Janeiro, Rio de Janeiro, RJ, Brasil
2Department of Periodontology, Metropolitan School of Dentistry, Barranquilla, Colombia
3Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil


Braz Dent J (1997) 8(1): 27-33 ISSN 0103-6440

| Introduction | Material and Methods | Results | Discussion | References |


The periodontal condition of 30 type II non-insulin-dependent diabetic patients (NIDDM) was evaluated and compared to that of 30 non-diabetic patients (age range, 30-77 years). Glycosylated hemoglobin and fasting glucose tests were used to measure the metabolic control of the patients, and the BANA (N-benzoyl-DL-arginine-naphthylamide) test was used to analyze subgingival microbiota. At the end of the study, the diabetic group was subdivided into three groups according to the degree of diabetic control: controlled, moderately controlled and poorly controlled. The non-parametric chi square test was used for statistical analyses: BANA test - no statistically significant differences were found between the two groups when analyzed as a whole. When the diabetic patients were subdivided and compared to the control group, significance was detected (P<0.01) in the total frequencies of the BANA scores (negative, moderately positive and positive: 1, 2, and 3, respectively) in the 5-, 6-, and 7-mm pockets and also in the 4-mm pockets in the poorly controlled group.


Key Words: periodontal disease progression, type II diabetes, probing depth, attachment loss, BANA test.


Introduction

Several studies have shown that bacterial plaque is the primary factor of periodontal disease and that its composition is different both among individuals and among different sites of the same individual (Loe et al., 1965; Theilade et al., 1966; Listgarten, 1976). Other studies have conceptualized periodontal disease as different forms of disease with similar clinical manifestations, meaning that different kinds of periodontal disease are caused by the different bacteria which compose the subgingival plaque (Slots and Genco, 1984; Dzink et al., 1985; Loesche et al., 1985).

Clinical and longitudinal evaluations of patients treated for periodontal disease and of patients who were not submitted to periodontal treatment led us to conclude that periodontal disease evolves through activity outbreaks, depending on factors such as type and pathogenicity of gingival sulcus bacterial plaque, host resistance and systemic involvement reflecting clinically as the presence of active areas (Haffajee et al., 1983).

Analyzing the enzymes produced by periodontopathogenic bacteria, Loesche (1986) and Loesche et al. (1987) broadened the perspective for bacteriological monitoring in the different phases of periodontal treatment, with practical applicability and low cost to the clinical periodontist.

Three of the most important periodontopathogenic bacteria, Bacteroides gingivalis (Porphyromonas gingivalis), Treponema denticola, Bacteroides forsythus, and a species of the genus Capnocytophaga hydrolyze the synthetic substrate N-benzoyl-DL-arginine-beta-naphthylamide (BANA). BANA showed a significant correlation with the number and ratio of spirochetes present in subgingival plaque samples from several sites (Loesche, 1986) and from individual sites (Loesche et al., 1987). These studies concluded that the BANA reaction can be used as an indicator of the spirochete level in individual sites of treated as well as non-treated patients.

In studies concerning the microbiota of diabetic patients with periodontitis, Shlossman et al. (1985) found that B. gingivalis is an important agent in periodontal disease of diabetic (NIDDM) Pima Indians. Studying the subgingival microflora of NIDDM patients with periodontitis with mild and normal tolerance to glucose, Zambon et al. (1988) reported a high prevalence of black pigmented Bacteroides suggesting that the ratio of B. gingivalis is higher than that for B. intermedius in NIDDM patients with periodontitis than in other groups. Microbiological and immunological data from this study suggest that B. intermedius, W. recta and B. gingivalis are important in the etiology of periodontitis in adult NIDDM patients.

Several studies, cited in Part I of this study (Novaes Jr. et al., 1996), were carried out concerning diabetes and the incidence of periodontal disease but the results were not unanimous as to the exact relationship of diabetes mellitus and the occurrence of oral disease.

The objectives of this study are to: 1) evaluate the use of the BANA test as an indicator of the presence of periodontopathogenic bacteria in periodontal pockets of type II NIDDM patients after 6 monthly evaluations; 2) establish a correlation between the probing depths and the results of the BANA test.


Material and Methods

The selection of the patients, the clinical methodology used to determine the probing depths in diabetic patients as well as in control patients, and the examination of the systemic condition are the same as in Part I of this study (Novaes Jr. et al., 1966). The BANA test was carried out monthly during the last six months of the one-year longitudinal study. The deepest pocket of each quadrant was selected for BANA analysis (Loesche et al., 1987).

Material from 720 sites of 30 diabetic patients and from 720 sites of 30 control patients was collected. During six months, 4 samples were collected from each patient (one in each quadrant, 120 samples) at the monthly examinations (total 720 samples). Bacterial plaque samples were collected after crown polishing of the previously selected areas, using a Gracey 5/6 (Hu-Friedy) sterile periodontal curette to reach the deepest area of the periodontal pocket (Loesche et al., 1987). To avoid contamination during sampling among the different sites of the same patient, two sterile curettes were used in each examination.

BANA hydrolysis reaction was carried out with Perioscan Oral-B cards (Redwood City, CA, USA). The subgingival plaque sample was immediately placed on a reactive matrix on the lower portion of the card. The precise site of the sample was indicated. The superior matrix was moistened with distilled water using a cotton swab and the card was folded in such a way that contact was established between the superior and inferior matrices. The card was then heated to 50-60°C for 15 min. The results were read using the reactive matrix according to Loesche et al. (1990).

Statistical analysis was carried out with the non-parametric chi square test.


Results

Diabetic Patients

Probing in 720 sites showed a prevalence of 34.4% for 5-mm pockets and 32.7% for 6-mm pockets. Probing depths of 4, 7, and 8 mm were observed less frequently (15.1%, 13.5%, and 4.3%, respectively) (Table 1).

When the frequency of probing depths of 4 to 8 mm and the BANA test scores (values from 1 to 3) were correlated, 41 sites were negative (score 1, 5.7%), 163 sites were moderately positive (score 2, 22.6%), and 516 sites were positive (score 3, 71.7%) (Table 1). The percent of negative reactions (BANA score 1) and moderately positive reactions (BANA score 2) tended to decrease when the probing depth increased, that is, they were inversely proportional to the increase in probing depth. However, when BANA was positive (score 3) the relationship was directly proportional (Figure 1A).

Control Patients

Probing of the 720 control sites showed a prevalence of 36.3% for 5-mm pockets and 33.2% for 6-mm pockets. Four- seven- and eight-mm deep pockets were observed less frequently (16.5%, 11.4% and 2.6%, respectively) (Table 1).

When periodontal probing depths (4-8 mm) and BANA scores (values 1 to 3) were correlated, 58 sites (8.0%) were negative (score 1), 164 sites (22.8%) were moderately positive (score 2) and 498 sites (69.2%) were positive (score 3) (Table 1). The percent of negative reactions (score 1) and moderately positive reactions (score 2) tended to decrease as probing depths increased. When BANA was positive (score 3) the relationship was directly proportional, that is, the percent of sites tended to increase as probing depth increased (Figure 1B).

Statistical analysis

Using the non-parametric chi square test to verify if there was a correlation between the diabetic (NIDDM) and the non-diabetic groups (control) in terms of different probing depths and BANA scores (negative, moderately positive and positive scores, 1, 2, and 3, respectively), no statistical significance was found (P>0.05) (Table 2). Correlating the subdivided diabetic groups (controlled, moderately controlled and poorly controlled) and the non-diabetic group (control) in terms of probing depths and BANA scores, no statistical significance was observed (P>0.05) in the 4-mm pockets of the controlled and moderately controlled groups; however, statistical significance (P<0.05) was detected for 4-mm pockets in the poorly controlled patients and 5-, 6-, and 7-mm pockets for all subdivisions (Table 3).


Discussion

BANA hydrolysis by periodontopathogenic bacteria such as Porphyromonas gingivalis, B. forsythus and T. denticola and the genus Capnocytophaga (Slots and Genco, 1984; Dzink et al., 1985; Loesche et al., 1985; Tanner and Bouldin, 1989; Omar et al., 1990) is an alternative method for diagnosis of periodontal diseases (Laughon et al., 1982a,b; Syed et al., 1984; Loesche et al., 1987, 1990a,b). Syed et al. (1984) reported that plaque from non-treated patients showed a high proportion of sites with enzymatic activity related to pocket depth. Loesche (1986) observed that 71.0% of the plaque sampled from pockets of non-treated patients were BANA positive and that sites similar to or larger than 7 mm showed 80.0% to 92.0% positive BANA reactions.

Our results for non-treated diabetic patients showed that 71.7% of the removed plaque was BANA positive; the 4-mm pockets were 79.6% positive and the 8-mm pockets were 100% BANA positive (Table 1). In the non-treated non-diabetic patients, 69.2% of the plaque was BANA positive; the 4-mm pockets were 47.9% BANA positive and, similarly, the 8-mm pockets were 100% BANA positive (Table 1). These results are quite similar to those of Loesche (1986) showing the importance of P. gingivalis, T. denticola and B. forsythus in the etiology of periodontal disease in non-diabetic patients. Our findings suggest that they are also important in diabetic patients.

The 4-, 5-, 6- and 7-mm periodontal sites of NIDDM patients showed 10.1, 6.1, 3.9 and 6.1% negative BANA results which could mean that the BANA negative microorganisms contribute to the observed periodontal alterations or that the percent of positive BANA microorganisms is under the limit of the BANA test. This hypothesis was presented by Loesche (1979) when comparing the sensitivity of the test with the results of ELISA, emphasizing false-positive results of 9%. In spite of the limitations of introducing the curette into some pockets to collect material, these percents were not significant, considering that the 8-mm pockets presented 100% positive results.

No statistically significant differences were observed (P>0.05) (Table 2) with the chi square test carried out with the total frequencies of the diabetic and control groups and BANA scores for 4-, 5-, 6-, and 7-mm pockets. Likewise no statistically significant differences (P>0.05) were found in the 4-mm pockets when considering the controlled and moderately controlled subdivisions of NIDDM patients and controls. However, in the poorly-controlled patients, statistical significance was detected at the 1% probability level (P<0.01). In the 5-, 6-, and 7-mm pockets for all groups, statistical significance was observed at P<0.01. This reinforces the suggestion that the metabolic degree of the diabetes is important (Table 3).

Shlossman et al. (1985) considered B. gingivalis an important etiological agent of periodontal disease in diabetic Pima Indians which agrees with the BANA test results which detected Porphyromonas gingivalis (formerly B. gingivalis), B. forsythus and T. denticola.

Microbiological data from the Zambon et al. study (1988) suggest that B. intermedius, W. recta and B. gingivalis (Porphyromonas gingivalis) are important in periodontal etiology of adult NIDDM patients. Bacterial infections represent a particular problem in the case of diabetes mellitus, since these patients often show deficiency in host defense and restoring mechanisms which can be intrinsic or secondary to lack of diabetes control. Subgingival microflora in diabetics with periodontitis is similar to that found in non-diabetic adults with severe periodontitis. Both groups showed high prevalence and a great proportion of anaerobic Gram-negative microorganisms including species of black pigmented Bacteroides, B. gingivalis and B. intermedius as well as W. recta. These species have also been previously involved in the etiology of periodontitis in the adult (Zambon et al., 1981; Moore et al., 1983), and associated with sites where active loss of periodontal attachment is observed.

Zambon et al. (1988) reported the importance of the Bacteroides species in periodontal infection which is consistent with previous studies suggesting that Bacteroides species can be important in the etiology of extra-oral infections in diabetic adults.

Our results confirm the etiological importance of Porphyromonas gingivalis (formerly B. gingivalis), and emphasize the possible participation of B. forsythus and T. denticola as etiological agents of periodontal disease in NIDDM patients.


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Correspondence: Arthur Belém Novaes Jr., Av. das Américas, 1155, Conj 1002, 22631-000 Rio de Janeiro, RJ, Brasil.


Accepted December 19, 1996
Electronic publication: September, 1997


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